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1521-0111/97/3/171–179$35.00 https://doi.org/10.1124/mol.119.118349 MOLECULAR Mol Pharmacol 97:171–179, March 2020 Copyright ª 2020 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

Bupropion Inhibits Type 3AB Heteromeric Channels at Clinically Relevant Concentrations

Antonia G. Stuebler and Michaela Jansen Department of Physiology and Molecular Biophysics and Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas Received September 25, 2019; accepted December 13, 2019 Downloaded from ABSTRACT , a Food and Administration–approved antide- bupropion was reversible and time-dependent. Of note, prein- pressant and aid, blocks and cubation with a low concentration of bupropion that mimics transporters and noncompetitively therapeutic drug conditions inhibits 5-HT–induced currents in 5- inhibits nicotinic and serotonin (5-HT) type 3A HT3A and 5-HT3AB receptors considerably. In summary, we receptors (5-HT3ARs). 5-HT3 receptors are pentameric - demonstrate that bupropion inhibits heteromeric 5-HT3ABRs as gated ion channels that regulate synaptic activity in the central well as homomeric 5-HT3ARs. This inhibition occurs at clinically molpharm.aspetjournals.org and peripheral nervous system, presynaptically and postsynap- relevant concentrations and may contribute to bupropion’s tically. In the present study, we examined and compared the clinical effects. effect of bupropion and its SIGNIFICANCE STATEMENT on mouse homomeric 5-HT3A and heteromeric 5-HT3AB recep- tors expressed in Xenopus laevis oocytes using two-electrode Clinical studies indicate that antagonizing serotonin (5-HT) voltage clamp experiments. Coapplication of bupropion or type 3AB (5-HT3AB) receptors in brain areas involved in mood hydroxybupropion with 5-HT dose dependently inhibited 5- regulation is successful in treating mood and anxiety disor- HT–induced currents in heteromeric 5-HT type 3AB receptors ders. Previously, bupropion was shown to be an antagonist at (5-HT3ABRs) (IC50 5 840 and 526 mM, respectively). The homopentameric 5-HT type 3A receptors. The present work

corresponding IC50s for bupropion and hydroxybupropion for provides novel insights into the pharmacological effects that at ASPET Journals on September 24, 2021 homomeric 5-HT3ARs were 10- and 5-fold lower, respectively bupropion exerts on heteromeric 5-HT3AB receptors, in partic- (87 and 113 mM). The inhibition of 5-HT3ARs and 5-HT3ABRs was ular when constantly present at low, clinically attainable non–use dependent and voltage independent, suggesting concentrations. The results advance the knowledge on the bupropion is not an open . The inhibition by clinical effects of bupropion as an .

Introduction peripheral nervous system (Thompson and Lummis, 2013) and are involved in virtually all brain functions (Hassaine The 5-hydroxytryptamine-3, or serotonin (5-HT) type 3, et al., 2014). is an ionotropic receptor and a member of the Cys- To date, five different 5-HT3 subunits have been identified loop family of pentameric ligand-gated ion channels, and (5-HT3A – 5-HT3E).Thefirstsubunittobecloned,5-HT3A thereby, differs from G-protein-coupled serotonin receptors (Maricq et al., 1991), is the only subunit among these that can (Thompson and Lummis, 2007). The 5-HT type 3 receptor (5- form functional homo-oligomeric receptors on the cell mem- HT3R) is similar in structure and function to other members of brane when expressed in Xenopus oocytes or cell lines (Hussy the pentameric ligand-gated family, including et al., 1994). Introduction of the 5-HT3B subunit yields cation-selective nicotinic acetylcholine (nACh) receptors functional heteromers with altered properties compared with (nAChRs) and anion-selective GABAA and receptors. the homo-oligomer and with heteromer function more closely Malfunction in these receptors has been linked to several resembling the functional responses observed in native neurologic disorders (Lemoine et al., 2012). Together, they tissues (Hussy et al., 1994; Davies et al., 1999). When compared are responsible for fast in the central and with 5-HT3A, the 5-HT type 3AB receptor (5-HT3ABR) differs in concentration-response curves, shows increased single- channel conductance and desensitization, and an altered Research reported in this publication was supported in part by the National Institute of Neurologic Disorders and Stroke of the National Institutes of current-voltage relationship (Davies et al., 1999; Dubin Health [R01 NS077114 (to M.J.)]. The content is solely the responsibility of the et al., 1999; Kelley et al., 2003b). authors and does not necessarily represent the official views of the National Institutes of Health. The 5-HT3R is widely distributed in the central and https://doi.org/10.1124/mol.119.118349. peripheral nervous systems and on extraneuronal cells, such

ABBREVIATIONS: 5-HT, serotonin; 5-HT3R, 5-HT type 3 receptor; 5-HT3AR, 5-HT type 3A receptor; 5-HT3ABR, 5-HT type 3AB receptor; Bup, bupropion; cRNA, complementary RNA; EC30, concentration that elicits approximately 30% of the maximal response; HydroB, hydroxybupropion; nACh, nicotinic acetylcholine; nAChR, nACh receptor; OR-2, oocyte ringer solution.

171 172 Stuebler and Jansen as monocytes, chondrocytes, T-cells, and synovial tissue (Fiebich et al., 2004). In the periphery, 5-HT3Rs are found in the autonomic, sensory, and enteric nervous systems (Faerber et al., 2007), where they are involved in regulating gastroin- testinal functions, such as motility, emesis, visceral percep- tion, and secretion (Niesler et al., 2003; Lummis, 2012). The highest density of 5-HT3Rs in the central nervous system is in the hindbrain, particularly the dorsal vagal complex involved in the vomiting reflex, and in limbic structures, notably the amygdala, hippocampus, nucleus accumbens, and striatum (Jones et al., 1992; Miyake et al., 1995). Substantial 5-HT3B expression was identified in the human brain with high levels in the amygdala, hippocampus, and the nucleus caudate (Dubin et al., 1999; Tzvetkov et al., 2007). A high amount of 5-HT3Rs are found on presynaptic nerve fibers (Nayak et al., 2000; Miquel et al., 2002), through which they can modulate the release of other , such as Downloaded from dopamine, cholecystokinin, GABA, substance P, and acetyl- (Chameau and van Hooft, 2006; Faerber et al., 2007). Fig. 1. Comparing 5-HT3ARs to 5-HT3ABRs. (A) Sample traces of 5-HT3A (black) and 5-HT3AB (green) at varying concentrations of 5-HT. (B) Owing to its involvement in many brain functions, the 5-HT3R Concentration-response curves show a higher potency of 5-HT at 5-HT3ARs represents an attractive therapeutic target. as compared with 5-HT3AB, as well as a steeper Hill slope. Parameters from 5 m 5 6 5 5-HT3R antagonists are used to effectively treat patients these curves: 5-HT3A:EC50 0.8 M, nH 2.53 0.58, n 5, 5-HT3AB:

5 m 5 6 5 molpharm.aspetjournals.org experiencing irritable bowel syndrome and chemotherapy-/ EC50 4.30 M, nH 1.04 0.02, n 8. Data are represented as the mean 6 S.D. (C) Direct comparison of 5-HT3A and 5-HT3AB inward current radiotherapy-induced and postoperative nausea and vomiting evoked by 1 mM 5-HT for 30 seconds. (Thompson and Lummis, 2007). Some (Choi et al., 2003; Eisensamer et al., 2003) and prepared in distilled water. Hydroxybupropion (100 mM, Toronto (Rammes et al., 2004) also antagonize 5-HT Rs, which, 3 Research Chemicals, Inc., North York, Canada) was dissolved in DMSO. together with other preclinical and clinical studies, suggests All solutions were made in oocyte ringer solution (OR-2) immediately the relevance of 5-HT3R antagonism for treating psychiat- before conducting experiments. ric disorders (Walstab et al., 2010; Bétry et al., 2011). We Molecular Biology. Complementary DNA encoding the mouse recently discovered that bupropion (Bup), another antide- 5-HT3AR (AAT37716) containing a V5-tag (GKPIPNPLLGLDSTQ) pressant, antagonizes 5-HT type 3A receptors (5-HT3ARs) close to the N-terminus (Jansen et al., 2008) and the mouse 5-HT3B at ASPET Journals on September 24, 2021 (Pandhare et al., 2017). receptor (NP_064670) in the pGEMHE vector were used for oocyte Bupropion was first approved as an “atypical” antidepressant expression (Reeves et al., 2001). Plasmids were linearized with the over 30 years ago, and today it is one of the most commonly NheI restriction and in vitro transcribed with the T7 RNA prescribed antidepressants. Despite its recognized clinical polymerase kit (mMESSAGE mMACHINE T7 Kit; Applied Bio- systems/Ambion, Austin, TX). Capped complementary RNA (cRNA) efficacy for both depression and smoking cessation, a com- was purified with the MEGAclear Kit (Applied Biosystems/Ambion) prehensive picture of how bupropion modulates neurotrans- and precipitated using 5 M ammonium acetate. cRNA dissolved in ’ mission is still emerging. Bupropion s therapeutic effect is nuclease-free water was stored at 280°C. thought to be mediated by the blocked reuptake of dopamine X. laevis Oocyte Preparation. Oocytes were isolated, enzymat- and norepinephrine (Stahl et al., 2004) and the noncompet- ically defolliculated, and stored as previously described (Goyal et al., itive inhibition of neuronal and muscular AChRs (Slemmer 2011). X. laevis frogs were handled and maintained following proce- et al., 2000). Most recently, the discovery that bupropion dures approved by the local animal welfare committee (Institutional Animal Care and Use Committee, IACUC no. 08014, PHS Assurance also noncompetitively inhibits 5-HT3ARs (Pandhare et al., 2017) raises the questions of whether this inhibition takes no. A 3056-01). In brief, the isolated oocytes were incubated with place at clinically relevant concentrations and if bupropion collagenase (collagenase from Clostridium histolyticum Type IA; Sigma-Aldrich) for 1 hour in OR-2 (115 mM NaCl, 2.5 mM KCl, also inhibits heteromeric members of the 5-HT3 family. 1.8 mM MgCl2, 10 mM HEPES, pH 7.5), which was followed by Therefore, we investigated the effect of bupropion and its extensive washing with OR-2. Oocytes were then rinsed three times major metabolite, hydroxybupropion (HydroB), on the func- with OR-2 1 2mMCa21 for 45 minutes each and maintained in tion of heteromeric 5-HT3ABRs as compared with the homo- standard oocyte saline medium [100 mM NaCl, 2 mM KCl, 1 mM meric 5-HT3ARs expressed in Xenopus oocytes. Here, we MgCl2,1.8mMCaCl2, 5 mM HEPES, pH 7.5, supplemented with demonstrate that 5-HT3ABRs, like 5-HT3ARs, are dose- 1% Antibiotic-Antimycotic (100, 10,000 U/ml of penicillin, dependently inhibited by bupropion and its metabolite. This 10,000 mg/ml of streptomycin and 25 mg/ml amphotericin B; Gibco, inhibition is voltage-independent and non–use dependent Thermo Fisher Scientific), 5% horse serum] for up to 7 days at 16°C. (i.e., affected by preincubation) and occurs at physiologically Oocytes were microinjected with 10 ng of in vitro synthesized cRNA m relevant concentrations. (200 ng/ l) using an automatic oocyte injector (Nanoject II; Drum- mond Scientific Co., Broomall, PA) up to 48 hours after isolation.

For optimal expression of the heteromeric 5-HT3ABR, the A and B Materials and Methods subunits were coinjected in a 1:3 ratio. This ratio has been shown to be optimal for 5-HT3ABR expression because a lower ratio results Materials. Stock of serotonin (2 mM 5-HT, serotonin creatinine in 5-HT3AR mimicked current response and a higher ratio would sulfate monohydrate; Acros Organics, New Jersey, NJ) and bupropion impact overall receptor expression (Thompson and Lummis, 2013; (100 mM, Toronto Research Chemicals, Inc., North York, Canada) were Corradi et al., 2015). Bupropion’s Effect on 5-HT3ABRs 173

Fig. 2. Bupropion’s antagonistic activity at homo- meric and heteromeric 5-HT3Rs. (A) Sample traces of oocytes expressing 5-HT3A or 5-HT3AB in re- sponse to 5-HT (∼EC30) alone and in combination with bupropion. 5-HT–evoked inward currents (gray, 5-HT3A 5 0.3 mM, 5-HT3AB 5 2 mM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of bupropion (5-HT3A:10–1000 mM, 5-HT3AB:30–4000 mM). (B) Currents were normalized to the control currents and yielded the following IC50 values: 5- HT3A:IC50 5 87.1 mM(nH 5 1.28 60.15, n 5 5, mean 6 S.D.) and 5-HT3AB:IC50 5 840 mM(nH 5 1.78 6

0.15, n 5 7, mean 6 S.D.). (C) Oocytes expressing Downloaded from 5-HT3A and 5-HT3AB did not elicit an inward current in response to bupropion alone. molpharm.aspetjournals.org

Electrophysiology. Two-electrode voltage clamp recordings were was fitted using the variable-slope sigmoidal dose response curve performed and analyzed using a TEV-200A amplifier (Dagan Instru- equations: ments, Minneapolis, MN), a Digidata 1440A data interface (Molecular Devices, Sunnyvale, CA), a MiniDigi 1B (Molecular Devices), and I 1 *100 5 (1a) ðLogEC50 2 XÞ*n the pClamp 10.7 software (Molecular Devices). Recordings were Imax 1 1 10 H conducted 1–4 days after microinjection. All experiments were – I 5 1 performed at room temperature (22 24°C) and at a holding *100 ð 2 Þ (1b) I 1 1 10 LogIC50 X *nH potential of 260 mV, unless otherwise stated. The oocytes were max held in a 250 mlchamberandperfusedwithOR-2usinggravity at ASPET Journals on September 24, 2021 Within these equations, Imax is the current activated at saturating 5- flow at an approximate rate of 5 ml/min. Drugs and serotonin were HT concentration, EC50 is the agonist concentration producing 50% of dissolved in the same solution and applied by gravity perfusion. the Imax,IC50 is the concentration of antagonist producing 50% in- The glass microelectrodes were filled with 3 M KCl and had hibition of Imax, X is the logarithm of agonist (eq. 1a) or antagonist (eq. a resistance of below 2 MV. /antagonists were applied 1b) concentration, and nH is the Hill coefficient. All figures and graphs until stable response or desensitization was observed to record were made in Origin and Adobe Illustrator CC 2018. maximal current responses. Data Analysis. All electrophysiological data were analyzed with pClamp, Origin (OriginLab Corporation, Northampton, MA) and GraphPad Prism 6 (GraphPad SoftwareSoft, La Jolla, CA). Data Results are represented as the mean 6 S.D., and maximal current Differentiating between 5-HT3AR and 5-HT3ABR. To induced by 5-HT was used as the normalizing standard (100% cur- evaluate the effect of bupropion and its major metabolite rent response) for other current responses in the same oocyte. hydroxybupropion (Fig. 1) on homomeric and heteromeric Statistical significance was determined with paired or unpaired t test (in Origin) with a cutoff for significance of 0.05 (*P # 0.05; 5-HT3 receptors, we expressed 5-HT3ARs and 5-HT3ABRs **P # 0.01; ***P # 0.001) or one-way ANOVA followed by [in a 1:3 A to B ratio (Thompson and Lummis, 2013; Corradi Dunnett’s multiple comparisons test (in Prism). The 5-HT (agonist et al., 2015)] in Xenopus oocytes. First, we substantiated stimulation—eq. 1a), bupropion, or hydroxybupropion (antagonist the obvious difference between the two receptor types inhibition—eq. 1b) concentration dependence on 5-HT3 currents (Fig. 1). The application of the agonist 5-HT to Xenopus

TABLE 1

EC50 (5-HT) and IC50 values (bupropion and hydroxybupropion) determined in X. laevis oocytes expressing 5-HT3ARs and 5- HT3ABRs. Data represented as mean 6 S.D. of n experiments. Statistical significance of A as compared with AB was determined with unpaired t test (*P # 0.05; **P # 0.01; ***P # 0.001). pEC50 and pIC50 are the negative logarithms of EC50 and IC50, respectively.

Agonist pEC50 (mM, Mean 6 S.D.) EC50 nH (Mean 6 S.D.) n

5-HT3AR 5-HT 6.10 6 0.02*** 0.80 2.53 6 0.58** 5 5-HT3ABR 5.38 6 0.03*** 4.30 1.04 6 0.02** 4

Antagonist pIC50 (mM, mean 6 S.D.) IC50 nH (Mean 6 S.D.) n

5-HT3AR Bupropion 4.06 6 0.05*** 87.1 1.28 6 0.15*** 5 5-HT3ABR 3.09 6 0.11*** 840 1.78 6 0.15*** 7 5-HT3AR Hydroxybupropion 3.95 6 0.10*** 113 1.17 6 0.14*** 5 5-HT3ABR 3.28 6 0.02*** 526 1.80 6 0.16*** 8 174 Stuebler and Jansen

Fig. 3. Hydroxybupropion is an antago- nist for 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5- HT3A or 5-HT3AB in response to 5-HT (∼EC30) alone and in combination with hydroxybupropion. 5-HT–evoked inward currents (gray, 5-HT3A 5 0.3 mM, 5-HT3AB 5 2 mM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increas- ing concentrations of hydroxybupropion (5-HT3A:10–1000 mM, 5-HT3AB:50–2000 mM). (B) Currents were normalized to the control currents and yielded the follow- ing IC50 values: 5-HT3A:IC50 5 113 mM Downloaded from (nH 5 1.17 6 0.15, n 5 5, mean 6 S.D.) and 5-HT3AB:IC50 5 526 mM(nH 5 1.80 6 0.16, n 5 8, mean 6 S.D.). (C) Oocytes expressing 5-HT3A and 5-HT3AB did not elicit an inward current in response to hydroxybupropion alone. molpharm.aspetjournals.org

oocytes expressing 5-HT3ARs (Fig. 1A, top trace) or 5-HT3ABRs Effect of Hydroxybupropion on 5-HT3AR and (Fig. 1A, bottom trace) elicits a rapid inward current with 5-HT3ABR. Hydroxybupropion, a major metabolite of bupro- a concentration-dependent amplitude. The currents resulting pion, is known to contribute to the biologic efficacy of the from a range of 5-HT concentrations were used to calculate the parent drug because it also inhibits dopamine/norepinephrine concentrations that produce a half-maximal response (Fig. 1B), transporters, nAChRs, and 5-HT3ARs (Bondarev et al., 2003; yielding EC50 values of 0.80 mM(n 5 5, Hill slope nH 5 2.53 6 Damaj et al., 2004; Pandhare et al., 2017). Similar to bupropion, at ASPET Journals on September 24, 2021 0.58) for the 5-HT3ARand4.30mM(n 5 8; nH 5 1.04 6 0.02) for hydroxybupropion inhibited 5-HT3ARs and 5-HT3ABRs dose- the 5-HT3ABR (Table 1). Our EC50 values are comparable to dependently when coapplied with 5-HT (Fig. 3A). The hydrox- those reported previously (Jansen et al., 2008; Lochner and ybupropion concentrations that reduced the 5-HT–evoked Lummis,2010),withtheheteromericreceptorshowing currents to 50% of the initial response were 113 mM(n 5 5, a right-shift in potency (Fig. 1B) and fast characteristic nH 5 1.17 6 0.14) for 5-HT3ARs and 526 mM(n 5 8, nH 5 1.80 6 desensitization kinetics (Fig. 1C). As reported in the 0.16) for 5-HT3ABRs (Fig. 3B; Table 1). Similar to bupropion, literature, the Hill coefficients are indicative of highly the potency of hydroxybupropion for 5-HT3ABRs was right- cooperative agonist sites for homopentameric channels and shifted, resulting in a higher IC50 valueascomparedto5-HT3ARs of a single site for heteropentameric channels (Thompson (unpaired t, t(11) 5 28.9, P 5 1.01 10211). Hydroxybupropion et al., 2011). did not elicit a response in 5-HT3A or 5-HT3AB expressing Effect of Bupropion on 5-HT3AR and 5-HT3ABR. 5-HT oocytes when applied alone (Fig. 3C). and a wide range of bupropion concentrations (A: 10–1000 mM; Effect of Preincubation with Bupropion and Hydrox- AB: 30–4000 mM) were coapplied to Xenopus oocytes expressing ybupropion on 5-HT3A and 5-HT3AB Receptors. Bupropion’s homomeric 5-HT3ARs (Fig. 2A, top) or heteromeric 5-HT3ABRs allosteric inhibition of 5-HT3ARs is not dependent on the (Fig. 2A, bottom) under two-electrode voltage clamp. 5-HT was opening of the receptor’s channel; it is non–use dependent applied at a concentration that elicits approximately 30% of the (Pandhare et al., 2017). To evaluate the extent of inhibi- maximal response (EC30) (5-HT3AR: 0.5 mM, 5-HT3ABR: 2 mM). tion evoked by preincubating oocytes expressing 5-HT3A and Both traces in Fig. 2A show representative current responses 5-HT3ABRs with bupropion or its metabolite, results were at 260 mV. The first inward current represents the control compared to the current amplitudes resulting from coappli- current evoked by 5-HT alone, and this is followed by cation of 5-HT and bupropion/hydroxybupropion. First, oocytes subsequent currents obtained by the coapplication of 5-HT were perfused with 5-HT (∼EC30, 5-HT3AR: 0.5 mM, 5-HT3ABR: (EC30) and increasing concentrations of bupropion, which dose- 2 mM) and bupropion (∼IC50, 5-HT3AR: 100 mM, 5-HT3ABR: dependently inhibited 5-HT-induced currents for 5-HT3A and 1 mM) to obtain the control current (Fig. 4A). Once a stable 5-HT3AB receptors. IC50 values were 87.1 mM(nH 5 1.28 6 response was achieved, a constant IC50 concentration of 0.15, n 5 5) and 840 mM(nH 5 1.78 6 0.15, n 5 7) for A bupropion was exposed to the receptors for exactly 5 min and AB, respectively (Fig. 2B; Table 1). Bupropion’s potency at before another coapplication of the same 5-HT and bupro- 5-HT3ABR was 10-fold lower when compared with 5-HT3AR pion solutions. Preincubation decreased the current ampli- 26 (unpaired t test, t(10) 5 8.49, P 5 6.99 10 ). Bupropion tude of 5-HT3ARs to 76.2% 6 7.16% (Fig. 4C, left panel) of alone did not elicit an inward current for either receptor control, consistent with previous findings (Pandhare et al., (Fig. ). 2017). On the contrary, under the same experimental conditions, Bupropion’s Effect on 5-HT3ABRs 175 the 5-HT3ABR was greatly affected by preincubation, which resulted in a current amplitude reduced to 35.5% 6 5.62% of the control current (Fig. 4C, right panel). Similar results were obtained from preincubation with hydroxybupropion (Fig. 4B). Compared with coapplication alone, preapplication resulted in a greater depression of current for 5-HT3ARs and 5-HT3ABRs with hydroxybupropion (Fig. 4C, 5-HT3AR: 93.0% 6 6.12% and 5-HT3ABR: 46.1% 6 4.95% of control current). Recovery Times for Bupropion Inhibition. Bupropion’s antagonistic effect on 5-HT–evoked inward currents has been shown to be reversible (Pandhare et al., 2017). To evaluate the recovery times of bupropion’s inhibition of 5-HT–induced currents at homomeric and heteromeric receptors, bupropion was applied to the oocytes for 60 s at a 400 mM concentration. For these experiments, the ∼EC50 concentration of 5-HT (gray bars, 5-HT3AR: 0.8 mM, 5-HT3ABR: 5.0 mM) was applied episodically after washing in between each application (∼2min). Downloaded from These agonist-induced currents led to minimal run-down, and the difference in current amplitudes was less than 10% (Fig. 5A). Sample traces of current recovery following bupropion appli- cation and removal are shown in Fig. 5B (left: black, 5-HT3AR, right: green, 5-HT3ABR). The first current response is the control current evoked by the agonist alone. Once a stable molpharm.aspetjournals.org current response was obtained, bupropion was applied Fig. 4. Non–use dependent allosteric inhibition of 5-HT Rs and 5- without the agonist for 60 s (magenta bars). Subsequently, 3A HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A (black, left the agonist was either applied immediately or after 10, 30, panel) and 5-HT3AB (green, right panel). The first 5-HT–evoked currents ∼ m or 60 s (Fig. 5B, top trace: 0 s, middle: 30 s, bottom: 60 s) were used for the control currents (gray bars, EC30, 5-HT3A: 0.5 M, 5- m after bupropion exposure. The largest decrease in current HT3AB:2 M) that were obtained by coapplication with bupropion (magenta bars, ∼IC50, 5-HT3A:100mM, 5-HT3AB: 1 mM). Following the amplitude for both receptors was immediately after the stable 5-HT response, bupropion (∼EC50) was perfused for 5 min before bupropion application, leaving 82.4% 6 3.08% and 38.4% 6 another coapplication of 5-HT and bupropion. (B) Same experimental ∼ 15.8% of the initial current for A and AB, respectively (Fig. 5C). design as in (A) but with hydroxybupropion (blue bars, IC50, 5-HT3A: 100 mM, 5-HT3AB:500mM). (C) Quantification of fractional inhibition of Rapid recovery of current amplitude was achieved by increasing currents when the oocyte was preincubated in bupropion (magenta) or at ASPET Journals on September 24, 2021 the wash times between bupropion and 5-HT applications. hydroxybupropion (blue) normalized to the control current (100%). Pre- incubation reduced current amplitudes for 5-HT (Bup: 76.1% 6 7.16%, 5-HT3ARs and 5-HT3ABRs both show time-dependent re- 3A n 5 5; HydroB: 93.0% 6 6.12%, n 5 6) and 5-HT3AB (Bup: 35.5% 6 5.62%, covery from bupropion inhibition with full reversal after n 5 6; HydroB: 46.1% 6 4.95%, n 5 4) as compared with coapplication. 71 minwashtime. Statistical significance was determined with paired t test (*P # 0.05; **P # Voltage-Independent Binding of Bupropion. To de- 0.01; ***P # 0.001), comparing coapplication (representing 100% of the 1 1 termine if bupropion binds to 5-HT Rs in a voltage-dependent current) to preapplication coapplication (Pre Co) with each drug 3 independently. Data are represented as the mean 6 S.D. manner, 5-HT–induced currents (∼EC50;5-HT3AR: 0.8 mM; 5-HT3ABR: 5.0 mM) were evoked in oocytes expressing 5- HT Rs and 5-HT Rs at two different holding potentials, exposed to three different 5-HT concentrations (0.5, 1.0, 3A 3AB 5.0 mM) in the absence of bupropion to obtain the initial 140 and 240 mV (Fig. 6A). First, the control current was obtained at positive and negative voltages before the coappli- current amplitudes (Fig. 7A, black, left panel: 5-HT3AR, cation of 5-HT and bupropion (∼IC ; 5-HT R: 100 mM; 5- green, right panel: 5-HT3ABR). Next, the oocytes were 50 3A continuously perfused with 20 mM bupropion, and the same HT R: 1 mM). Bupropion reduced the current amplitudes 3AB 5-HT concentrations were reapplied; the oocytes were preincu- of homomeric and heteromeric receptors at both voltages. The mean fractional block was recorded at each voltage and bated with bupropion for at least 2 minprior to 5-HT application (Fig. 7A, magenta bars indicating bupropion presence). The normalized to the control current (Fig. 6B; 5-HT R: 55.8% 6 3A results indicate that the continuous presence of a low concen- 0.11%, 59.8% 6 0.10%; 5-HT3ABR: 56.6% 6 0.04%, 59% 6 0.08%; 240 and 140 mV, respectively, n 5 4). For 5-HT R tration of bupropion in the bath solution partially inhibits 5- 3A – and 5-HT R, this fractional inhibition is similar at positive HT induced currents of 5-HT3ARs and 5-HT3ABRs at all 5-HT 3AB concentrations tested (Fig. 7B, paired t test, P # 0.05 or lower). and negative voltages (paired t test, 3A: t(3) 5 1.106, P 5 0.349; – ∼ 3AB: t(3) 5 0.291, P 5 0.790). Based on these results, inhibition Bupropion inhibited 5-HT induced currents by 18% for 5 ∼ of 5-HT–induced currents by bupropion is independent of 5-HT3ARs (n 4), whereas 5-HT3ABRs showed a 23% de- crease in current (n 5 5, Fig. 7B). voltage. Bupropion at Physiologic Concentrations and Its Effect on 5-HT3AR and 5-HT3ABR. To better understand Discussion the clinical significance of the bupropion-induced inhibition of 5-HT3Rs, 5-HT–induced currents were measured in the Our results, for the first time, demonstrate that bupropion presence of a clinically relevant bupropion concentration antagonizes heteromeric 5-HT3AB receptors and that the [∼20 mM (Schroeder, 1983; Vázquez-Gómez et al., 2014)]. kinetics of inhibition are distinct from 5-HT3A. Two-electrode First, oocytes expressing 5-HT3ARs and 5-HT3ABRs were voltage clamp experiments indicate that bupropion reversibly 176 Stuebler and Jansen

Fig. 5. Recovery times for bupropion. (A and B) Sample traces of bupropion application (magenta bar) and the recovery times for 5-HT3A (left panel, black) and 5-HT3AB (right panel, green). (A) In two-electrode voltage clamp experiments, oocytes expressing 5-HT3A and 5-HT3AB showed a stable response to repeated applications of 0.8 and 5 mM 5-HT at 260 mV, with an approximate wash time of 2 min. (B) The first 5-HT–evoked response represents the control current for the recovery experiment. Bupropion (400 mM) was applied alone for 60 s at 260 mV, followed by an immediate application of 5-HT. The gray and magenta bars represent the time of application of 5-HT and bupropion, respectively. Moving down the panel, the wash times after bupropion appli- cation were 0, 30, and 60 s. (C) Quantitative representation of current amplitudes and results in (B) (n 5 3). 5-HT3A was maximally reduced to 6 6

82.4% 3.08% and 5-HT3AB to 38.4% 15.8% of Downloaded from the control current when the agonist was applied immediately after bupropion, followed by a step- wise recovery. All currents could be recovered to ∼95% after ∼7.5-min wash. Statistical signifi- cance of each wash time as compared with the control current (the 5-HT–induced current re- sponse before exposure to Bup or HydroB) was determined with one-way ANOVA, Dunnett’s molpharm.aspetjournals.org multiple comparisons test (*P # 0.05; ***P # 0.001). Data are represented as the mean 6 S.D. inhibits 5-HT–induced currents of Xenopus oocytes expressing Similar to other noncompetitive antagonists [such as 5-HT3A and 5-HT3ABRs in a concentration-dependent manner, (Das and Dillon, 2003)], bupropion has a lower potency (∼10-fold) with inhibitory potencies of 87.1 mM[sameaspreviously at 5-HT3AB as compared to 5-HT3A receptors and, therefore, reported (Pandhare et al., 2017)] and 840 mM, respectively. couldbeusedtodiscriminatebetweenthesetworeceptors (Thompson and Lummis, 2013).

Similarly to bupropion, the metabolite inhibits nAChRs and at ASPET Journals on September 24, 2021 5-HT3ARs in a noncompetitive manner (Damaj et al., 2004; Pandhare et al., 2017) and also shares a comparable potency (unpaired t test, P value 5 0.06704) for the homomeric receptor [5-HT3AR: IC50 5 113 mM, similar to previously reported data (Pandhare et al., 2017)]. Hydroxybupropion exhibits a ∼4.5-fold shift in IC50 for the heteromeric channel (5-HT3ABR: IC50 5 526 mM). The estimated Hill slopes (nH values) for both bupropion and hydroxybupropion for both receptors were greater than unity (1.17–1.80), suggesting thepresenceofmultiplebindingsiteswithacooperative mechanism. The Hill coefficients for the 5-HT3ABRwere ∼1.4 times larger for both bupropion and hydroxybuporpion as compared with for the 5-HT3AR, which may indicate a concerted or cooperativity of bind- ing (Colquhoun, 1998). Bupropion-mediated inhibition of 5-HT3ARs is non–use de- pendent (Pandhare et al., 2017). In general, use-dependent block, or inhibition that would require a channel to be open to occur, is not influenced by preapplication. We evaluated the effect of a 5-min preincubation with bupropion and its metabolite hydroxybupropion on the homomeric and the heteromeric receptor (Fig. 4). Preincubation with antago-

Fig. 6. Voltage-independent block of 5-HT3–mediated currents by bupro- nistled to an increased inhibition in all cases when compared – – pion. (A) Sample traces of 5-HT3A and 5-HT3AB expressing oocytes (5- with coapplication, indicating that the block is non–use HT : left, black; 5-HT : right, green) in response to 5-HT (∼EC ; top 3A 3AB 50 dependent for both receptors. Our observation that bupropion’s and bottom traces, 5-HT3A:0.8mM; 5-HT3AB: 5.0 mM) in the absence and presence of bupropion (magenta traces, ∼IC50; 5-HT3A:100mM; 5-HT3AB: inhibition of 5-HT3Rs is voltage-independent additionally con- 1 mM) at different voltages. (B) Quantification of fractional inhibition, curs with it not acting as an open channel blocker (Slemmer 5 currents were normalized to the control currents at each voltage (n 4). et al., 2000; Choi et al., 2003; Gumilar et al., 2003). Similar Data are shown as mean 6 S.D. Statistical significance between the inhibition at positive and negative voltages was determined with paired results are shown with other antidepressants at 5-HT3Rs t test (*P # 0.05; **P # 0.01; ***P # 0.001). (Eisensamer et al., 2003) and with bupropion at nAChRs Bupropion’s Effect on 5-HT3ABRs 177 Downloaded from

Fig. 7. Bupropion at clinically achievable concentrations and its effect on 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A molpharm.aspetjournals.org (black, left panel) and 5-HT3AB (green, right panel) in response to 0.5, 1.0, and 5.0 mM 5-HT (gray bars) followed by the same concentrations coapplied with 20 mM bupropion. Following the initial exposure to the three 5-HT concentrations (control current), the oocytes were exposed to 20 mM bupropion for at least 2 min before coapplication with the agonist. (B) Quantitative representation of current amplitudes and results in (A) (A: n 5 4, AB: n 5 5). Data are shown as mean 6 S.D. Statistical significance between each 5-HT concentration without and with bupropion at the same 5-HT concentration was determined with paired t test (*P # 0.05; **P # 0.01; ***P # 0.001).

(e.g., a3b2, a4b2, a3b4) with predictions for an external currents. The largest reduction of current was observed with for bupropion (Slemmer et al., 2000; García- the shortest amount of wash time between the bupropion and Colunga et al., 2011). Considering different binding sites agonist applications (5-HT3AR: 82.4% 6 3.08%; 5-HT3ABR: within the family of AChRs (Pandhare et al., 2012), bupro- 38.4% 6 15.8% of the control current after 0-s wash). 5-HT3A at ASPET Journals on September 24, 2021 pion may have distinct binding sites in each channel (Arias and 5-HT3AB receptors show a time-dependent recovery from et al., 2009). bupropion’s inhibition, and their currents could be fully We saw a greater depression of current amplitudes when recovered after ∼7.5 min of washing. bupropion or its metabolite was preincubated as compared The clinical relevance of 5-HT3 inhibition by bupropion is with coapplication with 5-HT [Fig. 5, 5-HT3AR: 76.2% 6 currently unknown. Bupropion, but not its metabolites, con- 7.16%, 93.0% 6 6.12%; 5-HT3ABR: 35.5% 6 5.62%, 46.1% 6 centrates in many tissues with a brain to plasma ratio of 25:1 4.95% of control current, Bup and HydroB, respectively]. (Schroeder, 1983), which results in brain concentrations of During the preincubation experiments, bupropion binds and ∼20 mM (Vázquez-Gómez et al., 2014). Coapplication of 20 mM inhibits the receptor prior to the opening of the channel, bupropion with agonist minimally inhibits 5-HT–induced therefore presumably interacting with the closed channel and currents of 5-HT3ARs and does not affect HT3ABRs. On the potentially inhibiting the channel from opening (Choi et al., contrary, preincubation with bupropion has a drastic impact 2003; Arias et al., 2009). Consistent with other data, greater on its inhibitory effect (Fig. 7). Our results indicate that potencies of inhibition have been reported for bupropion and a preincubation time of 5 min with 20 mM bupropion is enough antidepressants on Cys-loop receptors in the resting to inhibit 5-HT3 receptors (5-HT3AR: ∼82.7% 5-HT3ABR: state than the open state (Choi et al., 2003; Gumilar and ∼74.9% of control current). Moreover, hydroxybupropion Bouzat, 2008; Arias et al., 2009). This phenomenon may be reaches ∼10-fold higher plasma concentrations in humans duetotheaccumulationofantidepressantsandantipsy- as compared with the parent drug (Findlay et al., 1981; chotics in the during preincubation, which Hsyu et al., 1997). With an average of ∼100 mM (based on may be important for the functional antagonistic effects of clinical data, test ID: FBUMT; Mayo Clinic, MN), hydroxybu- these drugs at the 5-HT3 receptor (Eisensamer et al., 2005). propion’s plasma concentrations are equivalent to 5-HT3AR’s Overall, this may indicate that bupropion has access to its IC50 value. Additionally, considering the increased inhibitory binding site(s) from the membrane environment. We find effect due to preincubation of 5-HT3Rs, we conclude that that inhibition after preincubation is more pronounced in bupropion and hydroxybupropion have the potential to inhibit the 5-HT3AB as compared with the 5-HT3A receptor. these receptors at clinically attainable concentrations. Bupropion’s inhibition of 5-HT–mediated currents is re- The comprehensive mechanism by which bupropion achieves versible after a substantial amount of washing. In this study, its therapeutic efficacy is multifactorial. At therapeutic dos- we investigated the time it takes for 5-HT3R to recover from ages, bupropion inhibits nACh receptors in the ventral tegmen- preincubation with bupropion at high concentrations (400 mM tal area, dorsal raphe nucleus , and interneurons in the Bup). Similar to our preincubation experiments, bupropion hippocampal CA1 area (Alkondon and Albuquerque, 2005; reduced 5-HT3AR currents but to a lesser extent than 5-HT3ABR Vázquez-Gómez et al., 2014). There, nAChRs can modulate 178 Stuebler and Jansen projections (Aznar et al., 2005; Chang et al., Chameau P and van Hooft JA (2006) Serotonin 5-HT(3) receptors in the central nervous system. Cell Tissue Res 326:573–581. 2011) and alter GABAergic transmission (Ji and Dani, Chang B, Daniele CA, Gallagher K, Madonia M, Mitchum RD, Barrett L, Vezina P, 2000), in turn increasing dopamine levels, contributing to and McGehee DS (2011) Nicotinic excitation of serotonergic projections from dorsal ’ raphe to the nucleus accumbens. J Neurophysiol 106:801–808. bupropion s antidepressant activity (Arias, 2009; Vázquez- Choi JS, Choi BH, Ahn HS, Kim MJ, Rhie DJ, Yoon SH, Min DS, Jo YH, Kim MS, Gómez et al., 2014). 5-HT3 receptors also show strong Sung KW, et al. 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Pandhare A, Pappu AS, Wilms H, Blanton MP, and Jansen M (2017) The antide- Walstab J, Rappold G, and Niesler B (2010) 5-HT(3) receptors: role in disease and Downloaded from pressant bupropion is a negative of serotonin type 3A recep- target of drugs. Pharmacol Ther 128:146–169. tors. Neuropharmacology 113 (Pt A):89–99. Rajkumar R and Mahesh R (2010) The auspicious role of the 5-HT3 receptor in depression: a probable neuronal target? J Psychopharmacol 24:455–469. Address correspondence to: Dr. Michaela Jansen, Department of Cell Rammes G, Eisensamer B, Ferrari U, Shapa M, Gimpl G, Gilling K, Parsons C, Physiology and Molecular Biophysics, Center for Membrane Protein Research, Riering K, Hapfelmeier G, Bondy B, et al. (2004) Antipsychotic drugs antagonize School of Medicine, Texas Tech University Health Sciences Center, 3601 4th human serotonin type 3 receptor currents in a noncompetitive manner. Mol Psy- St., Lubbock, TX 79430. E-mail: [email protected] chiatry 9:846–858, 818. molpharm.aspetjournals.org at ASPET Journals on September 24, 2021